Treatment of h-ras-driven tumors

ABSTRACT

The present disclosure describes a compositions and methods for treatment of Hras-driven cancers. Administration of a farnesyltransferase inhibitor, for example, tipifarnib, alone or in combination with a MEK inhibitor can reduce tumor size and tumor growth in cancers such as poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC)

CROSS-REFERENCE TO PRIORITY APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 15/305,778, filed on Oct. 21, 2016, which is a National StageApplication of PCT/US2015/027771, filed Apr. 27, 2015, which claims thepriority of U.S. Provisional Application No. 61/984,613, filed Apr. 25,2014. The entire disclosure of each of the prior applications isincorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to cancers associated withactivating mutations of H-Ras. More particularly, the present inventionrelates to treatment of those cancers by administration of afarnesyltransferase inhibitor (FTI).

BACKGROUND OF THE INVENTION

RAS-driven malignancies remain a major therapeutic challenge. Hras,KrasA, KrasB and Nras are plasma membrane GTPases that exist in anactive, GTP-bound or inactive, GDP-bound, state. Many human tumors havea predilection for mutations in one RAS gene family member. HRASmutations are less common overall, but they have a particularly highprevalence in cancers of the upper aerodigestive tract, skin, thyroidand urinary bladder.

All Ras isoforms are farnesylated. Farnesyl transferase inhibitors(FTIs) block the addition of an isoprenoid group to the C-terminalportion of Ras to prevent formation of active Ras. FTIs block Hrasfarnesylation, membrane localization and inhibit oncogenic Hras-drivencellular transformation in vitro and in vivo. However, in most clinicaltrials, FTIs showed no significant antitumor activity in patients withadvanced solid tumors such as lung, pancreatic and colon cancers, whichmainly harbor KRAS mutations or with acute myeloid leukemia, whichprimarily have mutations of NRAS.

Thus, a need exists for therapeutic agents able to inhibit growth andimprove outcome for patients with such cancers.

SUMMARY OF THE INVENTION

The invention provides a method for the targeted treatment of cancersassociated with a constitutively activating mutation of Hras. The methodcomprises administering to a subject whose tumor carries aconstitutively activating mutation such as Hras G12V, Hras Q61L or otherconstitutively activating mutation/substitution at codon 12, 13 or 61 ofHras, a therapeutically effective amount of a farnesyltransferaseinhibitor (FTI).

In one aspect, therefore, the invention relates to a method for thetreatment of a constitutively activating Hras mutation-driven cancersuch as thyroid cancer, salivary gland cancer, head and neck squamouscell carcinoma, bladder cancer, cervical cancer using a FTI, forexample, tipifarnib.

In a related aspect, the invention relates to a method for reducingtumor burden in a subject with a tumor that has a constitutivelyactivating mutation of Hras, the method comprising administering to thesubject a therapeutically effect amount of a farnesyltransferaseinhibitor (FTI).

In another related aspect, the invention relates to the use ofcombination therapy, that is, coadministration of tipifarnib andselumitinib for the treatment of a cancer associated with aconstitutively activating mutation of Hras.

In yet another related aspect, the invention relates to a method forreducing tumor burden in a subject with a tumor that has aconstitutively activating mutation of Hras by exposing the tumor to atherapeutically effective amount of an FTI

These, and other objects, features and advantages of this invention willbecome apparent from the following detailed description of the variousaspects of the invention taken in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show that Tpo-Cre/FR-HrasG12V/p53flox/flox mice developpoorly differentiated (1C top) and anaplastic (1C bottom) thyroidcancer. (A) is a schematic showing how mutant Hras was knocked into thenative mouse Hras1 gene locus in tandem with the wild-type copy (floxand replace). Upon the action of Cre recombinase, which is targeted tothe thyroid with the TPO promoter, the wild-type copy is excised andreplaced by HrasG12V, which is expressed physiologically under thecontrol of the endogenous Hras gene promoter. In addition, the p53 geneis knocked out by the excision of exons 2 through 10 in the presence ofCre-recombinase. (B) is a photo of a murine tumor using the abovedescribed genetic model (homozygous for both alleles). (C) hemotoxylinand eosin (H+E) sections of tumors collected fromTpo-Cre/FR-HrasG12V/p53flox/flox mice. Tumors are either poorlydifferentiated (top photo) or anaplastic (bottom photo; ratio 4:1).Poorly differentiated tumors are characterized by tightly packedcuboidal shaped cells with necrosis while anaplastic tumors are wellvascularized and have spindle shaped cells.

FIGS. 2A and B show the results of exposure of mouse cell lines fromtumor bearing Tpo-Cre/FR-HrasG12V/p53flox/flox mice tofarnesyltransferase inhibitors. Mouse cell lines were generated fromtumor bearing Tpo-Cre/FR-HrasG12V/p53flox/flox mice bycollagenase/dispase digestion and maintained in Coon's F-12 media withserum. After 15 passages, cells were plated in 1.5% serum and exposed toincreasing concentrations of indicated drug. (A) Western blots wereperformed for indicated proteins (antibodies from Cell Signaling withexception of Hras which was from Santa Cruz). Tipifarnib and lonafarnibdemonstrated dose dependent inhibition of the MAPK pathway signalingeffectors. (B) 6 day proliferation assays from the same mouse cell lineshowing dose-dependent inhibition of proliferation with tipifarnib andlonafarnib.

FIG. 3 is a schematic showing the design of the in vivo study oftipifarnib in Tpo-Cre/FR-Hras^(G121V)/p53^(flox/flox) mice. Mice weretreated for 14 days with tipifarnib 80 mg/kg BID by gavage. Tipifarnibwas prepared in 20% beta-cyclodextran. 3D ultrasound was performedbefore and after treatment to assess percent change in tumor volume.

FIG. 4 is a waterfall plot demonstrating percent change in thyroidvolume in mice treated with vehicle (blue) or 80 mg/kg tipifarnib BID.Tumors in each treatment group were sized matched at the beginning oftherapy. Substantial reduction in growth and tumor size was seen in mostcases treated with tipifarnib.

FIG. 5 is a waterfall plot of the same mice but with an additionalcohort of mice treated with lonafarnib added. Of note the lonafarnibtreated mice were not size matched to the vehicle group. Consistentinhibition of growth was observed between tipifarnib and lonafarnibgroups.

FIG. 6 shows body weight of vehicle- and tipifarnib-treated mice. Nodifferences were seen between the two groups. Overall, minimal toxicitywas observed after two weeks of treatment of tipifarnib 80 mg/kg BID.

FIG. 7 is a Kaplan-Meier survival curve showing survival ofTPO-Cre/Hras^(G12V+/+)/p53^(flox/flox) mice. These mice(heterozygous-squares and homozygous-triangles) have a high mortalitydue to disease burden as compared to mice with control (circles) orHrasG12V (inverted triangles) or p53 (diamonds) loss alone.

FIG. 8 shows the percent change in thyroid volume in Hras^(G12V+/+)/p53null mice with thyroid cancer following treatment with vehicle or 80mg/kg BID tipifarnib.

FIG. 9 is a Kaplan-Meier survival curve showing survival ofTPO-Cre/Hras^(G12V+/+)/p53^(flox/flox) mice treated with 80 mg/kg BIDtipifarnib.

FIG. 10 shows the results of targeting resistance to tipifarnib inTPO-Cre/Hras^(G12V+/+)/p53^(flox/flox) mice by combined treatment withAZD6244. A greater reduction in tumor size was observed in thecombination treatment (tipifarnib+AZD6244) for 14 days as compared toeither agent alone.

FIG. 11 shows that when treatment was extended for 28 days, increasedtumor growth was seen in the tipifarnib group, whereas mice treated withthe combination, tipifarnib+AZD6244, showed further reduction in tumorsize.

FIG. 12 shows that thyroid cancers from mice treated with thecombination showed a more profound decrease in Ki-67 staining and inexpression of Hmga2, a biomarker of the MAPK transcriptional output, ascompared to groups receiving vehicle, AZD6244 or tipifarnib alone.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents and other references (for example, thoselisted at the end of the specification) cited herein are incorporated byreference in their entirety into the present disclosure.

In accordance with the present disclosure there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explained indetail in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Terms used herein are intended to be interpreted consistently with themeaning known to those of skill in the art. A few terms as they would beknown in the art include the following.

As used herein, the terms “administering” and “administration” refer toany method of providing a composition disclosed herein to a subject orto bringing the composition into contact with the target tumor/cancer.Such methods are well known to those skilled in the art and include, butare not limited to, based on the location of the target tumor, oraladministration, transdermal administration, administration byinhalation, nasal administration, topical administration, intravaginaladministration, and parenteral administration, including injectableadministration such as intravenous administration, intra-arterialadministration, intramuscular administration, and subcutaneousadministration. Administration can be continuous or intermittent.

The compounds may be administered alone, or in combination with one ormore other compounds described herein, or in combination (i.e.co-administered) with one or more additional pharmaceutical agents.Combination therapy includes administration of a single pharmaceuticaldosage formulation containing one or more of the compounds and one ormore additional pharmaceutical agents, as well as administration of thecompounds and each additional pharmaceutical agent, in its own separatepharmaceutical dosage formulation. For example, one or more compoundsdescribed herein and one or more additional pharmaceutical agents, maybe administered to the patient together, in a single oral dosagecomposition having a fixed ratio of each active ingredient, such as atablet or capsule; or each agent may be administered in separate oraldosage formulations.

Where separate dosage formulations are used, the compounds and one ormore additional pharmaceutical agents may be administered at essentiallythe same time (e.g., concurrently) or at separately staggered times(e.g., sequentially).

As used herein, the term “effective amount” refers to an amount that issufficient to achieve the desired. For example, a “effective amount”refers to an amount that is sufficient to achieve the desiredtherapeutic result for example, will result in inhibition in the growthof tumor/cancer cells. In some embodiments, an effective amount willresult in the killing of tumor/cancer cells. The specifictherapeutically effective dose level for any particular subject willdepend upon a variety of factors including the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the subject; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs and/or radiation used incombination or coincidental with the specific compound employed and likefactors well known in the medical arts. For example, it is well withinthe skill of the art to start doses of a compound at levels lower thanthose required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days.

As used herein, the term “subject” refers to a target of administration,that is, an individual or organism, most often a patient in need oftreatment for an Hras mutation-driven tumor or cancer. The subject ofthe herein disclosed methods can be a human or non-human mammal.

The term “tumor burden” refers to the number of cancer cells, the sizeof a tumor, or the amount of cancer in the body.

As used herein, the terms “treatment” or “treating” relate to anytreatment of a condition associated with a presence of a mutantHras-driven cancer, including but not limited to prophylactic treatmentand therapeutic treatment. As such, the terms treatment or treatinginclude, but are not limited to: inhibiting the progression of thecondition; arresting the development of the condition; reducing theseverity of the condition; ameliorating or relieving symptoms associatedwith the condition; and causing a regression of the condition or one ormore of the symptoms associated with the condition of interest.

The presently-disclosed subject matter includes compositions and methodsfor targeting or producing an effect against cancer cells that harbor aconstitutively activating Hras mutation, including thyroid cancer.Compositions and methods of the presently-disclosed subject matter canhave utility in the treatment of thyroid cancer. Compositions of thepresently-disclosed subject matter include a farnesyl transferaseinhibitor (FTI), and a MEK inhibitor. In some embodiments, thecompositions are pharmaceutical compositions. Methods of thepresently-disclosed subject matter include administering an effectiveamount of a composition comprising a farnesyl transferase inhibitor(FTI) and in some embodiments, a MEK inhibitor to a subject. Thepresently-disclosed subject matter includes use of the compositionsdisclosed herein for the treatment of thyroid cancer.

The present invention is based on the observation that oncogenic Hraswith p53 loss results in anaplastic and poorly differentiated thyroidtumors in a mouse model and that tipifarnib inhibits mutant Hras inthese animals making tipifarnib an effective treatment for Hras-mutantcancers.

The tumor models used (anaplastic and poorly differentiated thyroidcancers) represent very aggressive malignancies with high proliferativerates. In other words, resistance to tipirfarnib that is observed invivo in these animals is accelerated compared to what would be seen inother cancer types. In those situations where resistance following FTItargeted therapy develops, addition of a MEK inhibitor, for example,AZD6244, to a farnesyltransferase inhibitor such as tipifarnib potentlyinhibits the MAPK pathway and tumor growth greater than either drugalone without additional toxicity.

Many human tumors have a predilection for mutations in one RAS genefamily member. However, there is no definitive explanation for thepredilection for individual RAS oncogenes in different tumor lineages.

All Ras isoforms are farnesylated. Farnesyl transferase inhibitors(FTIs) block the addition of an isoprenoid group to the C-terminalportion of Ras to prevent formation of active Ras. FTIs block Hrasfarnesylation, membrane localization, and inhibit oncogenic Hras-drivencellular transformation in vitro (19, 20) and in vivo (21). However, inmost clinical trials FTIs showed no significant antitumor activity inpatients with advanced solid tumors such as lung, pancreatic and coloncancers, which mainly harbor Kras mutations (22-24), or with acutemyeloid leukemia, which primarily have mutations of Nras (25). Therefractoriness to FTIs of RAS-driven cancers has been attributed tocompensatory geranylgeranylprenylation of Kras and Nras, which preservestheir membrane targeting and function (26-28). However, the Hrasselectivity of FTIs versus K- or N-ras-driven tumors has not beenextensively studied in cells or in a mouse model, and no trial with anFTI had been done exclusively in patients with Hras mutant tumors.

Activating mutations of Hras are found in 4-10% of advanced metastaticthyroid cancers and in a small fraction of other malignancies, such ashead and neck squamous carcinomas, salivary tumors, bladder cancer andothers. The mutations occur in, for example, codons 12, 13 and 61 ofHRAS. Examples of activating mutations of Hras include but are notlimited to G12V and Q61L.

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade totransmit signals from their receptors to regulate gene expression andprevent apoptosis. However, Ras signals through multiple effectorpathways and physiological activation of the Ras/Raf/MEK/Erk pathway isinfluenced by multiple mechanisms, and inhibitory molecules such as MAPKphosphatases that engage the pathway at different points to negativelyregulate signaling.

Determination of Tumor Hras Mutation Status

The following invention encompasses a method of treatment targetingHras-driven tumors, specifically those tumors known to have aconstitutively activating mutation of Hras.

Because treatment in accordance with this invention is targeted to suchcancers, knowledge of Hras status of the tumor prior to beginningtreatment can improve efficacy. In one embodiment, prior to beginningtherapy, DNA or RNA from cells from the tumor are assessed to determineHras mutation status to identify patients who are likely to benefit fromFTI/MEK inhibitor therapy. Mutation status is determined using standardsequencing methods known to those skilled in the art including, forexample, Sanger sequencing, next gen sequencing (NGS) etc., some ofwhich are described in more detail in the sequencing method reviewpublication found at the following url:illumina.com/content/dam/illumina-marketing/documents/products/research_reviews/sequencing-methods-review.pdf.

Tumors demonstrating a constitutively activating mutation at codon 12,13 or 61 of Hras would warrant treatment as described herein. In oneembodiment, the constitutively activating mutation is G12V of Hras; inone embodiment, the constitutively activating mutation is Q61L of Hras.

Farnesyl Transferase Inhibitors

Farnesyl transferase inhibitors (FTIs) are a class of compounds thattarget protein farnesyltransferase with the downstream effect ofpreventing the proper functioning of the Ras protein. FTIs are wellknown in the art and some, including Tipifarnib and Lonafarnib, havebeen fairly well characterized with respect to toxicity, bothhematological and non-hematological and pharmacokinetics.

Tipifarnib is a nonpeptidomimetic quinolinone that binds to and inhibitsthe enzyme, farnesyl transferase, thereby preventing the farnesylationof Ras isoforms. By inhibiting the farnesylation of these proteins, theagent prevents the activation of Ras oncogenes, inhibits cell growth,induces apoptosis and inhibits angiogenesis. Tipifarnib is commerciallyavailable from Jansen Pharmaceutica, NV under the name Zarnestra (formore details regarding tipifarnib, see U.S. Pat. Nos. 6,844,439,6,037,350, 6,150,377, and 6,169,096; the contents of each are herebyincorporated by reference in their entirety into the presentapplication.)

Lonafarnib is a synthetic tricyclic derivative of carboxamide that hasbeen shown to exhibit antineoplastic properties. Like tipifarnib,lonafarnib binds to and inhibits farnesyl transferase, the enzymeinvolved in the post-translational modification and activation of Rasproteins. Lonafarnib is commercially available under the brand nameSARASAR (Merck).

MEK Inhibitors

MEK (mitogen-activated protein kinase kinase) is a dual specificitykinase that phosphorylates both serine/threonine and tyrosine residues.MEK consists of two isoforms, MEK1 and MEK2, which in turn phosphorylateERK1 and ERK2.

MEK inhibitors are compounds that inhibit the MAPK kinase enzymes MEK1and/or MEK2 and therefore, can be used to affect the MAPK/ERK pathway.MEK inhibitors include but are not limited to AZD8330, Refametinib,Cobimetinib, E6201, binimetinib (MEK162), PD0325901, pimasertib,RO4987655, RO5126766, selumetinib, TAK-733, trametinib, GDC-0623, andWX-554.

Dosing

In one embodiment, tipifarnib is administered at a dose in the range of25 to 300 mg twice a day (bid). In another embodiment, tipifarnib isadministered at a dose in the range of 50 and 100 mg twice a day (bid).

In one embodiment, tipifarnib is administered orally to a subject inneed thereof at a dose of 300 mg bid for 21 consecutive days of a 28 daycycle with no drug administered for the remaining 7 days of the cycle.

In another embodiment, 400 mg is administered orally bid for twoconsecutive weeks followed by 1 week off (no drug.)

There are alternative dosing schedules that allow for higher drug dosesfor shorter periods of time, for example, 600 mg po bid for 1 weekfollowed by a break (no drug) for a week.

Dosing regimens may vary with the FTI used. One of skill in the artwould be able to determine the appropriate effective dose for aparticular FTI.

The dosages noted above may generally be administered for example once,twice or more per course of treatment, which may be repeated asnecessary as determined by the clinician.

In one embodiment, selumenitib is co-administered (see supra) at a dosein the range of 25 to 150 mg bid; in one embodiment in the range of 50to 100 mg bid; in one embodiment in the range of 70 to 80 mg bid.

EXAMPLES Experimental Animals and Tipifarnib Administration.

Mice with thyroid-specific activation of HrasG12V and p53 loss developaggressive thyroid tumors. Mice with thyroid-specific endogenousexpression of Hras-G12V+/+ and p53 loss (TPO-Cre/Hras-G12V+/+/p53f/f)(PMID: 11694875) were generated. These mice developed highly aggressivetumors between 6 weeks and 1 year of age (FIG. 1A, B). Immunostainingfor Ki-67 and pERK was increased in these tumors, consistent with ahighly proliferative tumor associated with activation of the MAP kinasepathway (data not shown). These mice have a high mortality due todisease burden as compared to mice with HrasG12V or p53 loss alone (FIG.7). Histologic examination demonstrated that anaplastic (ATC) or poorlydifferentiated thyroid cancers (PDTC) occur in these mice in a ratio ofapproximately 1:4, respectively (FIG. 1C).

Development and characterization of mouse cell lines from TPO-Cre,Hras-G12V+/+, p53f/f mice: In order to study this model in vitro, wedeveloped a mouse cell line (HP-ATC1) from an animal with ATC. The cellline was confirmed to harbor the HrasG12V mutation, and maintainedprimary tumor characteristics, including its spindle shape and therelative expression of E-cadherin and vimentin through serial passaging(×6-10).

The FTIs Tipifarnib and Lonafarnib block MAPK signaling and growth ofmouse Tpo-Cre, HRas-G12V+/+, p53f/f cell line: There are currently notherapies that directly target oncogenic forms of oncogenic Ras. Asfarnesyl transferase inhibitors have the potential to selectively targettumors driven by mutant Hras, we treated HP-ATC1 cells with increasingconcentrations of tipifarnib or lonafarnib, and found that they evoked adose-dependent inhibition of MAPK effector phosphorylation and ofproliferation (FIG. 2A, B).

Treatment of mice with Hras-G12V+/+/p53-null thyroid cancers with FTIsdemonstrates significant responses with resistance developing over time.The activity of these compounds in vivo was explored. Mice were treatedfor 2 weeks with tipifarnib or lonafarnib at 80 mg/kg twice daily (drugmixed in 20% beta-cyclodextran and given by gavage). Significantreduction in thyroid tumor volume (as measured by ultrasound) wasobserved compared to vehicle-treated mice (FIGS. 3,4,5). Treatment withFTIs was well tolerated in the mice with no significant differences inanimal weight between vehicle and drug at 14 days (FIG. 6). Next micewere treated with Tipifarnib or vehicle for an extended time course. Ofnote, for this experiment tumors in each group were size-matched at thetime of treatment initiation (FIG. 8). Mice treated with tipifarnib hadsignificantly less tumor growth as compared to vehicle over the 24-weektreatment period, which translated into a survival advantage (FIG. 9).However, in all tipifarnib treated animals, resistance ultimatelydeveloped as noted by increased tumor volume over time.

Targeting resistance to Tipifarnib in Tpo-Cre, Hras-G12V+/+, p53f/f miceby combined treatment with MEK inhibitor. Hras can signal throughnumerous effector pathways, including MAPK, PI3 kinase, RaIGDS andothers. Adaptive resistance could occur by reactivation of any of theseeffector pathways. Given that the MAPK pathway is central to thyroidcarcinogenesis, a MEK inhibitor (AZD6244) in combination with FTI wasused to prevent resistance. Mice were treated with either vehicle, 80mg/kg tipifarnib, 25 mg/kg AZD6244 or a combination of both drugs for 14days. Greater reduction in tumor size was observed in the combinationtreatment as compared to the other treatment conditions (FIG. 10). Whenthe treatment was extended for 28 days, increased tumor growth was seenin the tipifarnib group, whereas mice treated with the combinationshowed further reduction in tumor size (FIG. 11). Thyroid cancers frommice treated with the combination showed a more profound decrease inKi-67 staining and in expression of Hmga2, a biomarker of the MAPKtranscriptional output, as compared to the comparator groups (FIG. 12).The combination did not demonstrate any enhanced toxicity as opposed toeither drug alone.

Production of HrasG12V⁻p53⁻ mice (Tpo-Cre/FR-HrasG12V/p53^(flox/flox)mice). Mutant Hras was knocked into the native mouse Hras1 gene locus intandem with the wild-type copy (flox and replace). Upon the action ofCre recombinase, which is targeted to the thyroid with the TPO promoter,the wild-type copy is excised and replaced by HrasG12V, which isexpressed physiologically under the control of the endogenous Hras genepromoter. In addition, the p53 gene is knocked out by the excision ofexons 2 through 10 in the presence of Cre-recombinase.

Mouse cell lines were generated from tumor bearingTpo-Cre/FR-HrasG12V/p53flox/flox mice by collagenase/dispase digestionand maintained in Coon's F-12 media with serum. After 15 passages, cellswere plated in 1.5% serum and exposed to increasing concentrations ofindicated drug. Western blots were performed for indicated proteins(antibodies from Cell Signaling with exception of Hras which was fromSanta Cruz). Tipifarnib and lonafarnib demonstrated dose dependentinhibitor of the MAPK pathway signaling effectors (FIG. 2A). Six (6) dayproliferation assays from the same mouse cell line showed dose-dependentinhibition of proliferation with tipifarnib and lonafarnib (FIG. 2B).

Tissue Preparation, Histopathology and Immunohistochemistry.

Mice were killed by CO₂ asphyxiation. Normal and tumor tissue lysateswere prepared for extraction of RNA, DNA or protein as described (18).Histology was performed on H&E-stained formalin-fixed paraffin embeddedsections. Animal care and all procedures were approved by the MSKCCInstitutional Animal Care and Use Committee.

While several aspects of the present invention have been described anddepicted herein, alternative aspects may be effected by those skilled inthe art to accomplish the same objectives. Accordingly, it is intendedby the appended claims to cover all such alternative aspects as fallwithin the true spirit and scope of the invention.

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We claim:
 1. A method for reducing tumor burden in a subject with headand neck squamous cell carcinoma (HNSCC) comprising administering to thesubject a therapeutically effective amount of a farnesyltransferaseinhibitor (FTI), wherein the HNSCC comprises a constitutively activatingmutation of Hras.
 2. The method of claim 1, wherein thefarnesyltransferase inhibitor (FTI) is administered in combination witha MEK inhibitor.
 3. The method of claim 1, wherein the FTI is selectedfrom the group consisting of tipifarnib and lonafarnib.
 4. The method ofclaim 1, wherein the FTI is tipifarnib.
 5. The method of claim 2,wherein said MEK inhibitor is selected from the group consisting ofAZD8330, Refametinib, Cobimetinib, E6201, MEK162, PD0325901, pimasertib,RO4987655, RO5126766, selumetinib, TAK-733, trametinib, GDC-0623, andWX-554.
 6. The method of claim 1, wherein the constitutively activatingmutation comprises a mutation/substitution at codon 12, 13 or 61 ofHras.
 7. The method of claim 1, wherein the constitutively activatingmutation is Hras G12V.
 8. The method of claim 1, wherein theconstitutively activating mutation is Hras Q61L.
 9. The method of claim1, wherein tipifarnib is administered at a dose of 25-300 mg twice a day(bid).
 10. The method of claim 1, wherein tipifarnib is administered ata dose of 50-100 mg twice a day (bid).
 11. A method for the treatment ofa patient determined to have a head and neck squamous cell carcinoma(HNSCC) associated with a constitutively activating mutation of Hras,the method comprising administering to the patient a therapeuticallyeffective amount of a farnesyltransferase inhibitor (FTI).
 12. Themethod of claim 11, further comprising detecting a constitutivelyactivating mutation of Hras in a DNA or RNA sample from a cancer cellfrom the patient prior to administering the FTI.
 13. The method of claim11, wherein the FTI is administered in combination with a MEK inhibitor.14. The method of claim 11, wherein the constitutively activatingmutation comprises a mutation/substitution at codon 12, 13 or 61 ofHras.
 15. The method of claim 11, wherein the constitutively activatingmutation is Hras G12V.
 16. The method of claim 11, wherein theconstitutively activating mutation is Hras Q61L.
 17. A method for thetreatment of a head and neck squamous cell carcinoma (HNSCC) associatedwith a constitutively activating mutation of Hras in a patient in needthereof, comprising administering to the patient a therapeuticallyeffect amount of tipifarnib and selumetinib.
 18. The method of claim 17,wherein the constitutively activating mutation comprises amutation/substitution at codon 12, 13 or 61 of Hras.
 19. The method ofclaim 17, wherein the constitutively activating mutation is Hras G12V.20. The method of claim 17, wherein the constitutively activatingmutation is Hras Q61L.